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Ultra low strength electric field network-mediated ex vivo gene, protein and drug delivery in cells

The present application is related to U.S. Provisional Patent Application Ser. No. 60/663,562, filed on Mar. 19, 2005, which is incorporated herein by reference and to which priority is claimed pursuant to 35 USC 119.

1. Field of the Invention

The invention is in the field of methodologies to facilitate the ex vivo gene, protein or drug delivery in large quantity to various types of cells or cell clusters, such as islets, or various of tissues using an ultra low strength electric field network.

2. Description of the Prior Art

Electroporation is a technique involving the application of short duration, high intensity electric field pulses to cells or tissue. The electrical stimulus causes membrane destabilization and the subsequent formation of nanometer-sized pores. In this permeabilized state, the membrane can allow passage of DNA, enzymes, antibodies and other macromolecules into the cell. Electroporation holds potential not only in gene therapy, but also in other areas such as transdermal drug delivery and chemotherapy.

Since the early 1980s, electroporation has been used as a research tool for introducing DNA, RNA, proteins, other macromolecules, liposomes, latex beads, or whole virus particles into living cells. Electroporation efficiently introduces foreign genes into living cells, but the use of this technique had been restricted to the small quantity of suspensions of cell lines and primary cultures for basic research only, since the electric pulse are administered in a cuvette with a pair of needle type electrodes. No system has been established for the ex vivo low strength electroporation-mediated gene, protein or drug delivery to a large quantity of cells or a cell cluster, such as islet for therapeutic use.

Electroporation is commonly used for in vitro gene transfection, but limited work has been reported for in vivo gene transfer using a pair of needle or plate form electrodes in tumor, liver, myocardium in rodents. Most recently, an electroporation catheter has been used for delivery heparin to the rabbit arterial wall, and significantly increased the drug delivery efficiency. Most recently, we invented the systems for low strength electroporation-mediated in vivo gene, protein and drug delivery in organ and tissue of large animal and human (See U.S. Pat. No. 6,593,130 (2003) incorporated herein by reference. In that invention we also described a device for low strength electroporation-mediated ex vivo gene, protein and drug delivery in vessel of large animal and human.

No system has been described for the ex vivo low strength electroporation-mediated gene, protein or drug deliver into tissue or bioengineered tissue culture for therapeutic use. On the other hand, electric pulses with high electric field intensity can cause permanent cell membrane breakdown (cell lysis). According the best of knowledge now available, the voltage applied to any kind cells, whole embryo or embryonic heart in the cuvette setting must be as high as 200-1500 V/cm, and to any in vivo tissue using needle or plate form electrodes must be as high as 100-200 V/cm. Injury in such cases is a major concern, although it has never been well characterized.

One object of the invention is to establish the concept and applicable methodology for facilitate the ex vivo gene, protein or drug delivery to large quantities of various types of cells or cell clusters, such as islet, or various types of tissues using ultra low strength electroporation, which is defined in this specification as low strength electric field networking (LSEFN) strategies. The mechanism and nature of the bioelectric application in the present invention is only now being appreciated as being qualitatively different than prior art electroporation. Hence, to refer to the present bioelectric application as electroporation is misleading and inaccurate. Thus, hereinafter in this specification and in the medical field the present bioelectric application is referred to as low strength electric field networking (LSEFN). These bioengineered cells and tissues can then be transfused systemically, delivered or implanted into the various organs or tissue for the treatment of diseases.

The invention includes two components: 1) ex vivo gene, protein and drug delivery into the various of isolated cells mediated by the ultra low strength electric field; 2) ex vivo gene protein and drug delivery into cell clusters, such as islet, or whole embryos by the ultra low strength electric field; and 3) ex vivo gene, protein and drug delivery into the various types of cultured and bioengineered tissue using an ultra low strength electric field.

An LSEFN chamber is used which is shaped and sized to intimately contain the cells, cell clusters, or tissues in a transfusion chamber between opposing gas permeable membrane encapsulated electrode arrays across which low voltage LSEFN pulses are applied. The gas or gases introduced into the culture fluid through the gas permeable membrane can be chosen to optimize the specific metabolism and health required by the cell, cell clusters, or tissues. The high percentage of cell death, which is typical of prior art electroporation, is minimized or even avoided in the present application by the synergistic combination of low strength electric field network and optimal culture and gas environment for the cell, cell clusters, or tissues.

The illustrated invention is thus characterized by and has the advantages of: low voltage electro-permeabilization which results in less damage to the cell; dynamic electro-permeabilization, i.e. cells are moving in a static field, and rotating in a constant rate of buffer flow, therefore a constant temperature is maintained and heat damage to the cells is avoided thus allowing a long term LSEFN treatment as compared to the prior art; use of an electric array of very small electrodes to minimize heat and to use diffusing electric fields for providing a more nearly uniform average LSEFN exposure and transfusion into the cells; and a non-cuvette system which uses long exposure cell in a compact chamber to transfuse a large number of cells and to reintroduce them at a single time for large batch processing.

The invention is thus defined in its illustrated embodiment as a method of delivery of gene, protein or drug materials to macroscopic quantities of cells, cell clusters, or tissues comprising the steps of applying ex vivo LSFEN electric field to the cells, cell clusters, or tissues with an averaged field strength and an averaged electrical polarization of the LSEFN electric field; and systemically transfusing the gene, protein or drug materials into the cells, cell clusters, or tissues during LSEFN.

The method further comprises delivering in vivo the transfused cells, cell clusters, or tissues into organs or tissue.

The method further comprises the step of flowing a culture fluid to bathe the cells, cell clusters, or tissues during application of ex vivo an LSEFN electric field and during systemically transfusing the gene, protein or drug materials. The fluid may be used to culture the cells, cell clusters, or tissues. The flowing culture fluid easily mixes with the drug, protein or gene and increases the chance of the drug, protein or gene interaction with the cell membrane.

The step of applying ex vivo LSEFN electric field to the cells, cell clusters, or tissues comprises applying a pulsed DC electrical field with a predetermined burst repetition rate, each burst being separated by a predetermined rest period. The method step of applying ex vivo an LSEFN electric field to the cells, cell clusters, or tissues comprises applying an LSEFN electric field of less than 100 v/cm and preferably at approximately 10-1 V/cm or less. Regardless, of the numerically determined value of the LSEFN electric field, it is chosen at a magnitude which does not cause dielectric heating and biological damage to the cells, cell clusters, or tissues.

A flowing fluid maintains the temperature of the fluid substantially constant to avoid heat damage to the cells, cell clusters, or tissues in the LSEFN electric field.

The illustrated method of applying ex vivo an LSEFN electric field comprises the steps of disposing the cells, cell clusters, or tissues to the LSEFN electric field between at least one pair of electrodes across which the electric field is imposed. The electrodes are arranged and configured to provide a fringing field between them and being separated by a distance such that the cells, cell clusters, or tissues are primarily exposed to the fringing field so that the cells, cell clusters, or tissues are exposed to the averaged field strength and the averaged electrical polarization of the LSEFN electric field. An array of a pair of electrodes providing a positive and negative grid may be employed or a plurality of subarrays of various electrode elements employed in varied geometric arrangements.

The cells, cell clusters, or tissues are disposed into a chamber containing the cells, cell clusters, or tissues. The electrode arrays are disposed on or in the walls of the chamber, which walls intimately conform to the cells, cell clusters, or tissues subject to LSEFN, thereby providing the averaged field strength and the averaged electrical polarization of the LSEFN electric field. The LSEFN electric field is generated or applied using multiple arrays of a plurality of small electrodes to generate a pixilated fringing electric field. The size of the electrodes are chosen relative to the size of the cells, cell clusters, or tissues to provide an effective averaged field strength and the averaged electrical polarization of the LSEFN electric field at that scale.